Apparatus for mixing magnetic particles

ABSTRACT

An apparatus and method for carrying out the affinity separation of a target substance from a liquid test medium by mixing magnetic particles having surface immobilized ligand or receptor within the test medium to promote an affinity binding reaction between the ligand and the target substance. The test medium with the magnetic particles in a suitable container is removably mounted in an apparatus that creates a magnetic field gradient in the test medium. This magnetic gradient is used to induce the magnetic particles to move, thereby effecting mixing. The mixing is achieved either by movement of a magnet relative to a stationary container or movement of the container relative to a stationary magnet. In either case, the magnetic particles experience a continuous angular position change with the magnet. Concurrently with the relative angular movement between the magnet and the magnetic particles, the magnet is also moved along the length of the container causing the magnetic field gradient to sweep the entire length of the container. After the desired time, sufficient for the affinity reaction to occur, movement of the magnetic gradient is ended, whereby the magnetic particles are immobilized on the inside wall of the container nearest to the magnetic source. The remaining test medium is removed while the magnetic particles are retained on the wall of the container. The test medium or the particles may then be subjected to further processing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a division of copending application Ser. No.11/079,695, filed Mar. 15, 2005, which is a division of application Ser.No. 10/290,514, filed Oct. 8, 2002, now U.S. Pat. No. 6,884,357, whichis a continuation-in-part of application Ser. No. 09/771,665, filed onJan. 30, 2001, now U.S. Pat. No. 6,500,343, which is acontinuation-in-part of application Ser. No. 09/476,258, filed on Jan.3, 2000, now U.S. Pat. No. 6,228,268, and a continuation-in-part ofapplication Ser. No. 09/476,260, filed on Jan. 3, 2000, now U.S. Pat.No. 6,231,760,which is a division of application Ser. No. 08/902,164,filed Jul. 29, 1997, now U.S. Pat. No. 6,033,574, which is acontinuation-in-part of application Ser. No. 08/391,142, filed on Feb.21, 1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus and a method for mixingand separation of magnetic particles for the purpose of isolatingsubstances of interest from a nonmagnetic liquid test medium.

2. Description of Related Art

Magnetic separation of biomolecules and cells based on magneticparticles and employing biospecific affinity reactions is advantageousin terms of selectivity, simplicity, and speed. The technique has provedto be quite useful in analytical and preparative biotechnology and isnow being increasingly used for bioassays and isolation of targetsubstances such as cells, proteins, nucleic acid sequences and the like.

As used herein, the term “receptor” refers to any substance or group ofsubstances having biospecific binding affinity for a given ligand, tothe substantial exclusion of other substances. Among the receptorssusceptible to biospecific binding affinity reactions are antibodies(both monoclonal and polyclonol), antibody fragments, enzymes, nucleicacids, lectins and the like. The term “ligand” refers to substances suchas antigens, haptens, and various cell associated structures having atleast one characteristic determinant or epitope, which substances arecapable of being biospecifically recognized by and bound to a receptor.The term “target substance” refers to either member of a biospecificbinding affinity pair, i.e., a pair of substances or a substance and astructure exhibiting a mutual affinity of interaction, and includes suchthings as biological cells or cell components, biospecific ligands, andreceptors.

Affinity separation refers to known process techniques where a targetsubstance mixed with other substances in a liquid medium is bound to thesurface of a solid phase by a biospecific affinity binding reaction.Substances, which lack the specific molecule or structure of the targetsubstance, are not bound to the solid phase and can be removed to effectthe separation of the bound substance or vice versa. Small particles,particularly polymeric spherical particles as solid phase, have provedto be quite useful, as they can be conveniently coated withbiomolecules, provide a very high surface area, and give reasonablereaction kinetics. Separations of the particles containing bound targetsubstance (bound material) from the liquid medium (free material) may beaccomplished by filtration or gravitational effects, e.g., settling, orby centrifugation.

Separation of bound/free fractions is greatly simplified by employingmagnetizable particles, which allows the particle bound substance to beseparated by applying a magnetic field. Small magnetizable particles arewell known in the art, as is their use in the separations involvingimmunological and other biospecific affinity reactions. Smallmagnetizable particles generally fall into two broad categories. Thefirst category includes particles that are permanently magnetized, andthe second comprises particles that become magnetic only when subjectedto a magnetic field. The latter are referred to as paramagnetic orsuperparamagnetic particles and are usually preferred over thepermanently magnetized particles.

For many applications, the surface of paramagnetic particles is coatedwith a suitable ligand or receptor, such as antibodies, lectins, oligonucleotides, or other bioreactive molecules, which can selectively binda target substance in a mixture with other substances. Examples of smallmagnetic particles or beads are disclosed in U.S. Pat. No. 4,230,685,U.S. Pat. No. 4,554,088, and U.S. Pat. No. 4,628,037. The use ofparamagnetic particles is taught in publications, “Application ofMagnetic Beads in Bioassays,” by B., Haukanes, and C. Kvam,Bio/Technology, 11:60-63 (1993); “Removal of Neuroblastoma Cells fromBone Marrow with Monoclonal Antibodies Conjugated to MagneticMicrospheres” by J. G. Treleaven et. al. Lancet, Jan. 14, 1984, pages70-73; “Depletion of T Lymphocytes from Human Bone Marrow,” by F.Vartdal et. al. Transplantation, 43: 366-71 (1987); “Magnetic MonosizedPolymer Particles for Fast and Specific Fractionation of HumanMononuclear Cells,” by T. Lea et. al., Scandinavian Journal ofImmunology, 22: 207-16 (1985); and “Advances in BiomagneticSeparations,” (1994), M. Uhlen et. al. eds. Eaton Publishing Co.,Natick, Mass.

The magnetic separation process typically involves mixing the samplewith paramagnetic particles in a liquid medium to bind the targetsubstance by affinity reaction, and then separating the boundparticle/target complex from the sample medium by applying a magneticfield. All magnetic particles except those particles that are colloidal,settle in time. The liquid medium, therefore, must be agitated to somedegree to keep the particles suspended for a sufficient period of timeto allow the bioaffinity binding reaction to occur. Examples of knownagitation methods include shaking, swirling, rocking, rotation, orsimilar manipulations of a partially filled container. In some cases theaffinity bond between the target substance and the paramagneticparticles is relatively weak so as to be disrupted by strong turbulencein the liquid medium. In other cases biological target substances suchas cells, cellular fractions, and enzyme complexes are extremely fragileand will likewise be disrupted or denatured by excess turbulence.

Excess turbulence is just one of several significant drawbacks anddeficiencies of apparatus and methods used in the prior art forbiomagnetic separations. The specific configuration of a magneticseparation apparatus used for separating particle-bound target complexfrom the liquid medium will depend on the nature and size of magneticparticles. Paramagnetic particles in the size range of 0.1 to 300 μm arereadily removed by means of commercially available magnetic separationdevices. Examples of such magnetic separation devices are the Dynal MPCseries of separators manufactured by Dynal, Inc., Lake Success, N.Y.;and BioMag Separator series devices manufactured by PerSeptiveDiagnostics, Cambridge, Mass.; and a magnetic separator rack describedin U.S. Pat. No. 4,895,650. These devices employ permanent magnetslocated externally to a container holding a test medium and provide onlyfor separation. Mixing of the paramagnetic particles in the test mediumfor affinity binding reaction must be done separately. For example,Dynal MPC series of separators requires a separate mixing apparatus, aDynal Sample Mixer, for agitating the test media. The process must beactively monitored through various stages of mixing, washing, andseparation, and requires significant intervention from the operator.Accordingly, the efficiency of these devices is necessarily limited bythe skill and effectiveness of the operator.

U.S. Pat. No. 4,910,148 describes a device and method for separatingcancer cells from healthy cells. Immunoreactive paramagnetic particlesand bone marrow cells are mixed by agitating the liquid medium on arocking platform. Once the particles have bound to the cancer cells,they are separated from the liquid medium by magnets located externallyon the platform. Although such mixing minimizes the liquid turbulence,it does not provide an efficient degree of contact between the particlesand the target substance. Moreover, the utility of this device islimited to the separation of cells from relatively large sample volumes.

U.S. Pat. No. 5,238,812 describes a complicated device for rapid mixingto enhance bioaffinity binding reactions employing a U-tube-likestructure as mixer. The U-tube is rapidly rocked or rotated for 5 to 15seconds to mix the magnetic particles in the test medium, and then amagnet is brought in close proximity to the bottom of the U-tube toseparate the magnetic particles. As stated in the '812 patent, itsutility is limited to treating very small volumes (<1000 μl) of testmedium.

U.S. Pat. No. 5,336,760 describes a mixing and magnetic separationdevice comprising a chamber attached to a platform with one or moremagnets located close to the container and an intricate mechanism ofgears and motor to rotate the platform. Immuno-reactive paramagneticparticles are mixed in the test medium by first placing a stainlesssteel “keeper” between the chamber and the magnet to shield it from themagnetic field. Then the platform is rotated between vertical andhorizontal positions. The particles in the test medium are mixed byend-over-end movement of the chamber. Following the mixing, the “keeper”is removed so that the magnetic particles are captured by the exposedmagnetic field. Besides requiring a complicated mechanism, agitation ofthe liquid medium by end-over-end rotation does not mix relativelybuoyant particles efficiently, and the liquid turbulence will tend toshear off or damage the target substance.

U.S. Pat. No. 5,110,624, relates to a method of preparing magnetizableporous particles and describes a flow-through magnetically stabilizedfluidized bed (MSFB) column to isolate proteins from cell lysate. TheMSFB column is loosely packed with a bed of magnetizable particles andequipped with means of creating a stationary magnetic field that runsparallel to the flow of solution through the column. The particles aremaintained in a magnetically stabilized fluidized bed by adjusting therate of flow of the solution and the strength of the magnetic field.This is a complicated technique requiring precise adjustment of the flowrate and magnetic strength so that the combined effect of fluid velocityand magnetic attraction exactly counterbalances the effect of gravity onthe particles. Moreover, the design of MSFB is not optimized for usewith small test volumes, and cannot be made optimal for applicationssuch as bioassays or cell separations.

International patent application WO 91/09308 published Jun. 27, 1991discloses a separating and resuspending process and apparatus. Thisapplication teaches that rotation of a magnet around the containercontaining paramagnetic particles induces the particles to remain as acompact aggregate (in close proximity to the magnet source) and rollover one another. The application teaches that this method fails toproduce resuspension of the particles. The application WO 91/09308,discloses that the magnetic particles must be subjected to sequentialmagnetic fields situated opposite each other in order to effectresuspension. The application describes a device comprising a chamberlocated between two electromagnets, which are energized and de-energizedto aggregate the magnetic particles alternately at the two magnets. Theapplication teaches that alternately energizing and de-energizing thetwo electromagnets at a sufficiently rapid rate keeps the particlessuspended in the center of the chamber. This method limits movement ofthe particles to a relatively small distance, significantly reducing thecollision frequency between particles and the target substance,necessary for affinity binding which is a major reason for mixing theparamagnetic particles in the liquid medium. Moreover, a significantfraction of the particles, particularly particle-cell complexes mayescape the magnetic field by gravitational settling to the bottom ofchamber and will be lost during aspiration of the liquid mediumfollowing the aggregation step.

Japanese patent No. JP58193687 entitled Agitation And Separation OfMicroscopic Material is directed to separation of microorganisms bymixing magnetized ultra-fine magnetic wire with microorganismscontaining magnetic particles. The mixing is accomplished by a rotarymagnetic field, which also acts to separate the microorganisms after amixing step. This patent is concerned with separation of microorganismsthat contain internally ultra-fine magnetic particles. Suchmicroorganisms are well known in the art, a particular example beingmagneto spirillium, a bacteria known to synthesize ultra fine magneticparticles. Such microorganisms would not and cannot be used as magneticparticles for mixing and separation of a target species as envisioned bythe present invention. The Japanese patent's requirement forlinearly-connected ultra-fine magnetic particles refers to a wire whichis most likely used to create a high gradient magnetic field (HGMF) tocollect or precipitate the magnetite-containing bacteria over thesurface of these wires. Such a technique has no application to theprocess of affinity separation of a target substance from a liquid testmedium as envisioned by the present invention since it relies on themagnetic properties of the micro-organisms (the target substance itself)to effect a reaction.

The applicable known procedures have shortcomings, including therequirement for separate mechanically complex mixing mechanisms, as wellas various process constraints and inefficiencies. The present inventionprovides devices and methods for magnetic mixing and separation whichare of relatively simple construction and operation, which can beadapted to process large or small volumes of test liquid, and which canprocess multiple test samples simultaneously. Additionally, theinvention provides a single device for both mixing and separation and amethod which maximizes the mixing efficiency of the paramagneticparticles in the liquid medium without causing detrimental liquidturbulence, using an angular acceleration of at least 0.84radians/second/second (hereinafter referred as rads/s²).

SUMMARY OF THE INVENTION

According to the present invention, the affinity separation of a targetsubstance from a liquid test medium is carried out by mixing magneticparticles bearing surface immobilized ligands or receptors to promotespecific affinity binding reaction between the magnetic particles andthe target substance. The liquid test medium with the magnetic particlesin a suitable container is removably mounted in the apparatus of thepresent invention. In one preferred embodiment, a single magnetic fieldgradient is created in the liquid test medium. This gradient induces themagnetic particles to move towards the inside wall of the containernearest to the magnetic source. Relative movement between the magneticsource and the aggregating magnetic particles is started using apreferred angular acceleration of about 1.05 to about 4.19 rads/s² tomix the magnetic particles in the test medium and is continued for asufficient time to ensure optimum binding of the target substance byaffinity reaction. In addition, concurrently with the relative movement,the magnetic source may be moved from one end of the container to theother thereby effectively scanning along the length of the container bythe magnetic field gradient. When the relative movement between themagnet and the magnetic particles is stopped, the magnetic particles areimmobilized as a relatively compact aggregate on the inside wall of thecontainer nearest to the magnetic source. The test medium may then beremoved while the magnetic particles are retained on the wall of thecontainer and may be subjected to further processing, as desired.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the present invention, which are believed tobe novel, are set forth with particularity in the appended claims. Thepresent invention, both as to its organization and manner of operation,together with further objects and advantages, may best be understood byreference to the following description, taken in connection with theaccompanying drawings, wherein:

FIG. 1 shows a perspective view of a preferred embodiment of theinvention, which includes a stationary magnet placed next to a mobilecontainer partially filled with a liquid test medium containing magneticparticles.

FIG. 2 shows a perspective of an alternate preferred embodiment of theinvention, which includes a mobile magnet placed next to a stationarycontainer partially filled with a liquid test medium containing magneticparticles.

FIG. 3 shows a perspective of another preferred embodiment of theinvention, which includes a row of mobile magnets placed next tocorresponding stationary containers, which are rotationally displaced bya common mechanism.

FIG. 4 shows a perspective of another preferred embodiment of theinvention, which includes a row of stationary magnets placed next tocorresponding rotatable containers, which are rotated by a commonmechanism.

FIGS. 5 a, 5 b, 5 c, 5 d, 5 e and 5 f schematically illustrate the stepsof a method according to the invention for mixing and separation of atarget substance employing magnetic particles using the preferredembodiment of FIG. 2.

FIG. 6 shows a perspective view of a magnetic field gradient cavity in atest liquid medium according to the invention caused by one permanentmagnet placed close to the container.

FIG. 7 shows a perspective view of a magnetic field gradient cavity in aliquid test medium according to the invention caused by two magnetsplaced at the opposite sides of the container.

FIG. 8 shows a perspective view of multiple magnetic field gradientcavities in a liquid test medium according to the invention caused by avertical array of six permanent magnets placed close to the container.

FIG. 9 shows a perspective view of multiple magnetic field gradientcavities in a liquid test medium according to the invention caused bytwo vertical arrays of permanent magnets placed at the opposite sides ofthe container.

FIG. 10 a shows a perspective top view of another preferred embodimentof the invention, which includes two electromagnets placed at oppositesides of the container.

FIG. 10 b shows a perspective top view of yet another preferredembodiment of the invention, which includes a ring of electromagnetssurrounding the container.

FIGS. 11 a and 11 b schematically illustrate the magnetic field linescreated in a container by two magnets placed on opposite sides of thecontainer.

FIG. 12 shows a perspective view of yet another alternate preferredembodiment of the invention which includes a row of magnets mounted on avertically mobile assembly moveable by a linear drive mechanism andwhich can be positioned by a sliding mechanism at a desired distancefrom the corresponding rotatable containers, which are rotated by acommon mechanism.

FIG. 13A shows an isometric view of yet another alternate preferredembodiment of the invention, which includes magnets mounted in twoconcentric circular arrangement on a static plate and the containersinserted in a circular pattern of holes in a rotor plate which byrotation alternately positions the opposite sides of the containers infront of the each circular array of magnets.

FIG. 13B shows a view corresponding to FIG. 13A without the rotor plateand showing concentrically arranged magnets in a staggered pattern andthe containers positioned in front of each magnet.

FIG. 13C shows an exploded view corresponding to FIG. 1A, and showingvarious components of the embodiment.

FIG. 14A shows yet another alternate preferred embodiment of a inventionwhich includes containers inserted in a linear pattern the holes of astatic plate and a linear array of magnets mounted in a linear patternon a moveable support plate which by horizontally moving back and forthalternately brings the magnets on the opposite sides of the containers.

FIG. 14B shows the partially cut away view corresponding to FIG. 14A andshowing the details of the linear sliding mechanism of a groove in theside plate and the moveable support plate.

FIG. 14C shows a view corresponding to FIG. 14A without the containerholding plate and showing linear arrays of staggered magnets and thecorresponding containers.

FIG. 14D shows a partial top view corresponding to FIG. 14A showing thepositions of magnets in the three rows of magnet arrays and thecorresponding position of the containers.

FIG. 15 shows an isometric view of yet another preferred embodiment ofthe invention for the 96-well microplate format containers and lineararrays of magnets mounted on two independently moveable supportstructures, which by alternate movements in the vertical direction bringthe magnets between the wells of the 96-well microplate.

FIG. 15A shows an exploded view corresponding to FIG. 15 and showingdifferent parts of the device and their respective positions.

FIG. 15B shows a side view corresponding to FIG. 15A illustrating therelative positions of magnets on the two support structures.

FIG. 15C shows a top view corresponding to FIG. 15 with the positions ofthe magnets between the wells of the 96-well microplate brought in bythe vertical motion of one support structure.

FIG. 15D shows a top view corresponding to FIG. 15 with the positions ofthe magnets between the wells of the 96-well microplate brought in bythe vertical motion of second support structure.

FIG. 16A shows an isometric view of yet another preferred embodiment ofthe invention, which includes a container, mounted in a fixed holder andpositioned between two concentric rotor discs fixed to a rotating shaft.FIG. 16B shows a side view, corresponding to FIG. 16A and shows thepositions of the container and the two magnets mounted on the two rotorswhich upon rotation alternately positions the magnets on the oppositesides of the container.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor for carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the principles of the present invention are definedherein specifically to provide an apparatus and method for mixing andseparating samples containing paramagnetic particles, which maximize themixing efficiency of the particles without causing significant liquidmedium turbulence.

The invention permits rapid, efficient, and clean separation of a targetsubstance from test media and is particularly useful in the affinitymagnetic separations of organic, biochemical, or cellular components ofinterest from, for example, assay reaction mixtures, cell cultures, bodyfluids and the like. The invention includes a novel mixing systemwherein the magnetic particles are mixed within a relatively motionlesstest liquid by magnetic means disposed external to the container holdingthe test liquid. The invention also includes an apparatus and methodwherein magnetic particles while mixing and confined in a magnetic zoneare concurrently linearly displaced to scan large volumes of test mediumfor affinity separation with a small concentration of magneticparticles. The invention provides an apparatus in which both theprocesses of mixing and separation are carried out by a common magneticmeans disposed in a single apparatus, thereby making it simpler and morepractical to use.

The apparatus of the invention comprises at least one container forholding a test medium, external magnetic means to generate a magneticfield gradient within the test medium, and means for creating amagnetically induced movement of the magnetic particles within the testmedium. The apparatus of the invention may also include a linear motionmechanism to move the magnetic means for scanning large volume of theliquid test medium. The container for performing the described mixingand separation is preferably of cylindrical configuration, made of anonmagnetic material such as glass or plastic. Preferably, the containerhas at least one opening for receiving the test medium containing themagnetic particles.

The magnetic means may comprise one or more permanent or electromagnetsdisposed externally to the container for generating magnetic fieldgradients within the liquid test medium. In a preferred embodiment, themagnet is a permanent magnet of a rare earth alloy such as anisotropicsintered materials composed of neodymium-iron-boron or samarium-cobalt.The magnet is disposed external to the container so as to define amagnetic field gradient cavity in a desired cross-section of the testmedium. The term cavity is employed because the magnetic field gradientacts to confine or concentrate the magnetic particles much as if theywere enclosed within a cavity. The magnetic field strength in the cavityis normally stronger at a part of the internal surface of the containercloser to the magnet (locus of magnetic force) than it is elsewhere inthe cavity and becomes negligible outside the cavity. As a result,magnetic particles in the test medium near this locus are subject toconsiderably greater magnetic force than those farther from it and tendto aggregate as a relatively compact mass on the inner surface of thelateral wall of the container closest to magnetic means. As theparticles are all clustered in the vicinity of the magnetic means, theyalso tend to stick to each other by nonmagnetic forces of compressionand surface tension. The degree of compression in the aggregatedparticles depends on the field strength of magnetic means and isparticularly relevant in the case of particles with diameters of a fewmicrons, such as are usually employed in affinity separation. Suchcompacted particles can remain aggregated even after the removal of themagnetic field and usually require vigorous shaking of the test mediumto re-disperse. A carefully balanced magnetic field strength in the testmedium will pull the particles out of suspension into an aggregate, butwill not be so strong as to overly compress the aggregate. According tothe present invention, a desired magnetic field strength within themagnetic field cavity of the test medium may be created by appropriatelyadjusting the distance between the magnet and the container. Theapparatus of the invention provides means for adjusting the distancebetween a magnet and the container.

In certain preferred embodiments, two magnets may be located on theopposite sides of the container, preferably with similar magnetic polesfacing each other, to distort the magnetic flux lines and generate twomagnetic field gradients and two loci of magnetic force forming in onecavity. Such an arrangement is particularly useful for agitatingmagnetic particles, as described below. In a particularly advantageousarrangement, an assembly comprising a vertical array of magnets ispositioned exterior to the container to create multiple magnetic fieldgradient cavities within desired cross-sections of the test medium.

The present invention provides two methods for agitating and mixing themagnetic particles within the test medium while maintaining the testmedium substantially motionless with respect to the container. Bothmethods are based on changing the relative angular position betweenmagnetic means and the aggregated particles on the inside surface of thecontainer at an angular acceleration of at least 0.84 rad/s² andpreferably between about 1.05 to 4.19 rads/s². The first methodcomprises rotating the container with respect to a stationary magnet.The magnetic field gradient cavity defined by the magnet in thisinstance is hence stationary. At an angular acceleration of about 0.83to about 4.19 rads/s², the test medium is not agitated and rotates withthe container. The second method comprises rotating a magnet about astationary container. The magnetic field gradient cavity defined by themagnet in this instance is rotating. It may be noted that using eithermethod causes a change in the angular position between the aggregatedparticles within the container and the magnet.

As the relative angular position between the container and the magnet isdisplaced at an angular acceleration of at least 0.84 rad/s² andpreferably between about 1.05 to about 4.19 rad/s², the aggregated massof particles move with the wall of the container to a position of weakermagnetic field. At this position, the stronger magnetic field in thevicinity of the magnetic means begins to pull off the particles from theaggregated mass, the trajectories of the particles being pulled offdepends on the angular position of the aggregated mass and magnet. Asthe particles are pulled, they move and form chains of particles, due tothe induced magnetic dipole on the particles by the applied magneticfield. As the chains accelerate towards the magnet, fluid drag forcecauses them to break creating a cloud of magnetic particles in the fluidmedium. At a constant angular acceleration of either the container ormagnet, the relative angular position between the magnet and theinternal surface of the container bearing the aggregated particlesrecedes continuously and causes the particles to move ceaselessly inangular trajectories within the test medium thereby enabling there-suspension and mixing of magnetic particles. The parameter, angularacceleration, is important in the mixing of the magnetic particles asdescribed in the present invention. Applicant has found that at angularaccelerations below 0.84 rad/s², the aggregated mass of particles on theinside wall of the container do not move sufficiently rapidly toovercome the strong magnetic field in the vicinity of the magneticmeans, resulting in a rolling mass of aggregated particles. Angularacceleration between about 1.05 to 4.19 rad/s² permits the aggregatedmass of particles to move away with the wall of the container to aposition of weaker magnetic field thereby effecting mixing as describedabove.

As regards particles it should be noted that the force pulling amagnetic particle through a fluid medium is the product of its magneticsaturation and field gradient and the viscous force opposing particlemotion, which is governed by Stokes Law. The displacement of particletrajectories in a continuous manner is based on the action ofmagnetomotive force acting at a continuously changing angle between themagnet and the paramagnetic particles which results in a mixing processwithout fluid turbulence. Furthermore, this mixing process significantlyincreases the collision frequency between the particles and targetspecies thereby enhancing the efficiency of the affinity bindingreaction.

A suitable angular acceleration can be calculated on the basis of radiusof the container, forces of gravity, buoyancy, fluid friction andmagnetic field strength. However, for a given set of parameters, theintensity of the magnetic field or fields and the appropriate angularacceleration will be modulated experimentally. It should be noted thattoo high acceleration will not allow the particles sufficient time todetach from the aggregated mass and particles will be spread over thecircumference of the inner wall of the container. Similarly, too slowacceleration such as about 0.10 to 0.21 rads/s² will produce a rollingmass of the aggregated particles. In both cases, re-suspension andmixing of the particles will be prevented. The field strength in themagnetic field cavity of the test medium must also be balanced so as toallow the aggregated particles to move with the wall of the container.It will be appreciated that a fixed magnet position is inconvenient whenthe desired particle size may vary considerably. In such situations, itis advantageous to be able to adjust the distance between the magnet andthe container to create the optimum field strength in the magnetic fieldcavity of the fluid medium.

Although angular acceleration in the sense described above can beobtained by continuous rotation and provides satisfactory mixing ofmagnetic particles, in certain situations it is advantageous to providea step-wise change of a predetermined angular position. For example, therelative angular position may be changed to 90 or 180 degrees in asingle step at a significantly higher angular acceleration than isuseful in continuous rotation method. Such steps may be repeated morethan once, provided a suitable time delay is imposed between such steps.Applicant has found that angular acceleration as high as 300 rads/s² mayapplied when the relative angular position is changed to 180° in asingle step mode provided a time delay of at least 0.5 second is imposedbetween subsequent steps. Such repeated step-wise change of apredetermined angular position provides a very efficient mixing ofmagnetic particles. It should be noted that the selection of suitableangular acceleration is particularly important in the present inventionwith respect to the said mixing operation. In general, a specificangular acceleration, to ensure re-suspension and mixing, will depend onthe size, density and magnetic susceptibility of the particles, thecross sectional diameter of the container, the density and viscosity ofthe fluid test medium and the strength of the magnetic field. Although atheoretical calculation of such an angular acceleration is possible, fora given set of parameters an appropriate angular acceleration will bedetermined experimentally. The International patent application WO91/09308 cited earlier, discloses that rotating the magnet around thecontainer fails to produce resuspension as the particles remainaggregated. The cited WO 91/09308 is silent on the importance of angularacceleration with regard to resuspending and mixing process of theaggregated particles. As shown in example 1 (see later), Applicant hasexperimentally verified this teaching of the cited WO 91/09308 and hasfound that at an angular acceleration at or below 0.21 rads/s² theparticles roll over one another and remain substantially aggregated. Asa consequence the affinity binding reaction between particles and targetspecies would be seriously hindered and the isolation efficiency wouldbe reduced to almost zero. The experiments described in Example 2clearly demonstrate the effect of angular acceleration on thepurification efficiency. Applicant has found that angular accelerationabove 0.84 rad/s² and preferably between about 1.05 to 4.19 rad/s² isnecessary to provide useful mixing and resuspension of magneticparticles required in affinity binding reaction for the isolation of adesired target species. By recognizing the importance of angularacceleration the present Application overcomes the problem of non-mixingdisclosed in the prior art cited above.

In certain situations, re-suspension and mixing of magnetic particlesmay be improved by creating a magnetic field gradient in which themagnetic flux lines are distorted by providing two magnets placed on theopposite sides of the container with similar magnetic poles facing eachother as shown in FIG. 11 a. The magnetic field lines generated by thetwo magnets are mutually repulsive and the cavity is characterized byhaving two zones with corresponding loci of high magnetic attraction anda small region in the center (neutral zone) where there is virtually nomagnetic field. Since this neutral zone is very small, the random motionof magnetic particles caused by Brownian, gravitational, thermal, andlike causes will tend to push most of the magnetic particles into eitherof the two magnetic field cavities. In a dynamic situation where therelative angular position between the magnet and the container iscontinuously changing, opposing magnetic flux lines cause the magneticparticles to disperse and mix more efficiently than in the case of asingle magnet. However, when two magnets are of opposite poles, as shownin FIG. 11 b, the magnetic field lines are mutually attractive and thecavity is characterized by having two relatively small magnetic fieldswith corresponding loci of high magnetic attractions and a large regionin the center (neutral zone) where there is virtually no magnetic field.Such an arrangement may be of use in certain situations.

The separation of magnetic particles from the liquid test medium inaccordance with the invention is effected by stopping the rotation ofeither the magnet or the container to terminate the agitation of themagnetic particles. In the stationary position between magnet andaggregated particles, the magnetic particles within the magnetic fieldgradient in the fluid medium are attracted to and immobilized at theinside wall of the container nearest to the magnet.

The need for a reliable and readily automated method for resuspendingand mixing the aggregated magnetic particles without causing fluidturbulence has not been satisfactorily addressed. Applicant's inventionutilizes a new principle of mixing which has allowed, for the firsttime, integration of a simplified mixing and separation process into asingle device.

The present invention provides many advantages over the prior artdevices for affinity magnetic separation. The mixing of the presentinvention provides a high rate of contact between the affinity surfaceof the magnetic particles and the target substance, thereby enhancingthe affinity bonding, without causing fluid turbulence. As aconsequence, the hydrodynamic shear forces remain low and will notaffect the affinity bond between particle and target substance complexor prevent denaturation, or other damage to the target substance. Theprocess of the present invention can be used for sample volumes aslittle as 100 μL and can be scaled up to process sample volumes inexcess of 100 mL. The present invention is particularly useful for theisolation of human rare cells required in various cell therapies as itpermits a level of operating efficiency, which has not been achievablebefore this.

The purity and yield of the target substance obtained by a particularaffinity magnetic separation is largely determined by the mixing processemployed to promote the affinity binding reaction between the targetsubstance and the surface of the magnetic particles. The bindingreactions require a close contact between the affinity surface and thetarget substance. The rate of the reaction largely depends on thecollision frequency between the two entities and the rate of surfacerenewal of the magnetic particles. The surface renewal is the process ofremoving the thin layer of media at the affinity surface and exchangingit with fresh media from the bulk. The hydrodynamic shear force at theaffinity surface, therefore, must be carefully balanced so that it issufficient to remove the thin layer of media without disrupting theaffinity bonds. This has been difficult to achieve by past mixingmethods based on agitating the fluid medium. The present invention,however, provides a high collision frequency and a substantiallybalanced shear force by magnetically inducing a controlled movement ofthe magnetic particles in an essentially motionless fluid medium.

In affinity magnetic separation, the particle concentration is,typically, much greater than the target substance to enhance the yieldof the target substance. This is particularly important in the isolationof rare cell types such as mammalian hemopoietic cells where a particleto cell ratio of 20:1 may be required to obtain a desired isolationefficiency. In such applications, magnetic beads of uniform sizedistribution are required. The high cost of these beads are widelyappreciated. The ability to isolate highly purified stem cells may servein the treatment of lymphomas and leukemias as well as other neoplasticconditions. However, for the isolation of human stem cells, processingof large sample volumes is required. Such a process consumes largequantities of magnetic beads. Thus there is a need to reduce theconcentration of magnetic beads without sacrificing the required highpurity and yield. One embodiment of the present invention is capable oftreating large sample volumes by relatively small concentrations ofparamagnetic particles by combining a vertically moving magnet along thelength of the container while the container is rotating.

The mixing and separation process of the present invention haveparticular utility in various laboratory and clinical proceduresinvolving biospecific affinity binding reactions for separations. Insuch procedures, magnetic particles are used which have their surfacecoated with one member of a specific affinity binding pair, i.e. ligandor receptor, capable of specifically binding a substance of interest inthe test medium.

Such biospecific affinity binding reactions may be employed for thedetermination or isolation of a wide range of target substances inbiological samples. Examples of target substances are, cells, cellcomponents, cell subpopulations (both eukaryotic and prokaryotic),bacteria, viruses, parasites, antigens, specific antibodies, nucleicacid sequences and the like. The apparatus and method of the inventionmay be used to carry out immunospecific cell separations for theanalysis or isolation of cells including, by way of example: tumor cellsfrom bone marrow; T-lymphocytes from peripheral blood or bone marrow;lymphocyte subsets, such as CD2, CD4, CD8, and CD34 from peripheralblood, monocytes; granulocytes and other cell types. The removal ordepletion of various cell types may be carried out in a similar manner.The invention may be also be used in the separation or analysis ofvarious bacteria or parasites from food products, culture media, bodyfluids and the like. Similarly, the apparatus and method of the presentinvention may be used in: bioassays including immunoassays and nucleicacid probe assays; isolation and detection of DNA and mRNA directly fromcrude cell lysate; and isolation and detection of proteins.

The magnetic particles preferred for the practice of the invention arenoncolloidal paramagnetic or superparamagnetic particles. Such magneticparticles are typically of polymeric material containing a small amountof ferromagnetic substance such as iron-based oxides, e.g., magnetite,transition metals, or rare earth elements, which causes them to becaptured by a magnetic field. The paramagnetic particles useful forpracticing the invention should provide for an adequate binding surfacecapacity for the adsorption or covalent coupling of one member of aspecific affinity binding pair, i.e. ligand or receptor. The preferreddiameter of a particle is typically in the range between 0.1 to 15 μm.Suitable paramagnetic particles are commercially available from DynalInc. of Lake Success, N.Y.; PerSeptive Diagnostics, Inc., of Cambridge,Mass.; and Cortex Biochem Inc., of San Leandro, Calif. Particularlypreferred particles are spherical and of uniform size between about 1and 5 μm in diameter, and contain magnetizable material evenly dispersedthroughout. Such particles may be obtained from Dynal under theidentification numbers M-280 and M-450 by Dynal Inc. These beads arecoated with a thin shell of polystyrene, which provides a definedsurface for the immobilization of various ligands or receptors. Suchimmobilization may be carried out by any one of many well-knowntechniques; techniques employing either physical adsorption or covalentcoupling chemistry are preferred.

Depending on the radius of container, size of magnetic particle and itsferromagnetic content, and other experimental parameters, a suitablemagnetic field gradient may be estimated by the magnetic circuitanalysis method well known in the magnet art. Appropriate magnetic fieldgradients may be generated by one or more permanent magnet(s) orelectromagnet(s). Permanent magnets are generally preferred for use inlaboratory-scale operations and for automated devices employed inclinical diagnostics. A permanent magnet assembly may include soft ironpieces to enhance or modify the magnetic flux lines over a given areainside the container. In some situations, a magnet assembly comprisingtwo soft iron pieces separately attached to north and south poles of themagnet provide a more thorough and uniform mixing. However, larger scaledevices or automated devices such as those employed in pharmaceutical orindustrial production can be more advantageously produced usingelectromagnets, since the field gradients can be more easily alteredunder automatic control to affect various processing steps.

Permanent magnets for practicing the invention preferably have a surfacefield strength sufficient to attract a majority of the magneticparticles. Permanent magnets of rare earth alloys having a surface fieldstrength in the range of several hundred Gauss to several kilo-Gauss arepreferred. High energy permanent magnets made from Neodymium-Boron orSamarium-Cobalt magnets and characterized by BHmax (maximum energyproduct) in the range of about 25 to 50 MGOe (megaGauss Oersted) areparticularly preferred. Such magnets may be obtained from InternationalMagnaproducts Inc., of Valparaiso, Ind., and many other commercialsources. Preferably the permanent magnets have a rectangularcross-section and may be glued or fixed by mechanical means to anonmagnetic holding support to form a permanent magnet assembly. Theassembly may include a ferromagnetic harness to house the magnet ormagnets and to intensify and focus the magnetic field. The magnets arepreferably oriented with their magnetic lines of force perpendicular tothe vertical axis of the container. Alternate cross-sectional shapes,orientations, and magnetic pole orientation with respect to thecontainer are also envisioned.

Generally the permanent magnet assembly is placed in close proximity tothe container without the magnet extending to the bottom of thecontainer. The distance between each magnet and the container shown inFIGS. 1 through 6 and 12 is adjustable between about 1 mm to about 20 mmto create a desired magnetic field strength within the magnetic fieldcavity of the test medium. The apparatus shown in these figures includesa means for adjusting the distance between each magnet assembly and thecontainer. An adjusting means is shown in FIG. 12. Lateral (orhorizontal) movement of magnets is provided by a linear motionmechanism. Linear motion mechanisms are well known in the art. A simplelinear motion mechanism comprises a slider with a rectangular notch orgroove, riding on rail with corresponding rectangular shape. Such linearmotion mechanisms exist in common furniture drawers. Multiple rails canbe provided, as well as ball bearings and rollers if desired. A gearrack and pinion mechanism comprising of a rectangular gear teeth bar(rack) and a mating gear teeth pulley is advantageous when accuracy inthe distance between magnet assembly and the container is desired.Suitable gear racks and pinions are available from Designatronics Inc.,2101 Jericho Turnpike, New Hyde Park, N.Y. 11042-5416. Lateral movementof magnet assembly can also be changed by attaching it to anelectromagnetic actuator or plunger and such lateral movement may besynchronized with the rotary motion of the container or magnet assembly.Electromagnetic actuators or plungers are also well known in motioncontrol art. While FIG. 12 shows six cylindrical containers, obviouslythe number can be increased, or decreased to one. FIG. 12 further showsvertical movements of magnets driven by screw 116. Obviously structuresshown in FIG. 1 and FIG. 4 can be moved by FIG. 12 mechanisms. Forinstance, the stationary containers of FIGS. 2 and 3 or the stationarymagnet of FIG. 1 could be made movable using a screw mechanism, orsimilar mechanical means, like the one shown in FIG. 12. The magnetposition can also be changed by fastening the magnet assembly at adesired position by various male and female fasteners.

Depending on the size and magnetic susceptibility of the particles andthe field strength of the magnets and cross-section diameter of thecontainer, the appropriate distance will be determined experimentally.The field strength created in the magnet field cavities should becarefully balanced so that it is sufficient to pull the particles out ofsuspension, aggregate the particles on the side of the container, andallow the aggregated particles to move with the wall of the container.However, the magnet may be moved closer to the container, as discussed,to increase the field strength in order to separate the particles fromthe liquid test medium. In certain situations involving the processingof a plurality of containers, it may be advantageous to place thepermanent magnet assembly between containers or between rows ofcontainers so that one single permanent magnet assembly can be used togenerate a magnetic field cavity in the two containers in its vicinity.

Specifically, in order to move the stationary magnet along the verticalaxis of the moving container, as shown in FIG. 1, the solid support 2may be mechanically fastened to a carriage on a linear slide mechanismsimilar to the one (122) shown in FIG. 12. The apparatus and methodshown in FIG. 1 is simply a one-container variation of FIG. 12.

In the case of moving magnets as shown in FIGS. 2 and 3 where thecontainer(s) remains stationary, the rotating magnets may besimultaneously moved along the vertical axis of the stationary containerby providing a hole in the rotatable support 22 (FIG. 2), support 22being mounted on a hollow shaft electric motor which is available fromEAD Motors, 1 Progress Drive, Dover, N.H., U.S.A., and the motor itselfbeing mounted on a motorized linear slide mechanism such as shown inFIG. 12. The internal diameters of the hole in support 22 (FIG. 2) andthe hollow shaft of the electric motor will be larger than the container29 outer diameter. Hence the hollow shaft rotates and passes freely fromone end of the container to the other. Similarly, in the case of FIG. 3,appropriately positioned holes may be provided on the support 35. Theinternal diameters of the holes in support 35 are sufficiently large sothat the support 35 rotates freely around the containers and the lengthof the support shafts 34A and 34B (FIG. 3) mounted on pulleys 38A and38B will be sufficiently long to accommodate the length of thecontainers. The entire rotation assembly as shown in FIG. 3 may bemounted on a motorized linear slide mechanism such as shown in FIG. 12.

FIG. 1 illustrates an apparatus for mixing and separating magneticparticles according to the present invention, which includes a magnet 1next to a container 3. The magnet 1 is adjustably fixed to a solidsupport 2 without extending to the container's bottom end. The magnet 1is preferably movable with respect to the container 3 to adjust themagnetic field strength as desired. In the preferred embodiment, thecontainer 3 is a test tube used for holding a liquid medium 8 withmagnetic particles 9 shown as small dots located in the medium. If themagnet 1 is a permanent magnet, it preferably comprises a rare earthcomposite type such as Neodymium-Iron-Boron or Samarium-Cobalt and has asurface field strength of about 200 Gauss to 5 kilo Gauss, which issufficient to attract the magnetic particles in the size range of about0.1 μm to 10 μm. The permanent magnet employed has dimensions andgeometries that define a magnetic field cavity of a desired fieldstrength having a desired cross-section within the liquid test medium 8in the container 3. An electromagnet of comparable field strength may beused for the magnet 1.

The container 3 with the liquid medium 8 and the magnetic particles 9 isremovably placed in a vertical position in a holder 5. The holder 5 isfixed to a rotating shaft 4, which is in turn attached to a variablespeed electric motor 6. The holder 5 has vertical slits 7 which areelastic, to receive and firmly grip the container 3 in a verticalposition. The electric motor 6 rotates the container 3 causing therelative angular position of the aggregating magnetic particles 9 in thecontainer 3 with respect to the magnet 1 to be continuously altered,thereby inducing the magnetic particles 9 to move within the cavity ofthe magnetic field gradient defined within the test medium 8.

The motor 6 may be an electric step motor instead of a continuousrotation motor to provide a step-wise change of a predetermined distancein the relative angular position. Step movements of a predefined anglemay be repeated more than once, and if desired, with time delays from afraction of a second to tens of seconds between each step. Such steprotation would be accomplished by an electronic motor control that iswell known in the art. Other means for effecting step-wise motion andtime-delays well known in the electro-mechanical art could also be used.

The container 3 when rotated continuously is rotated from a restingposition to a speed, preferably between about 50 and 200 rpm in lessthan one second. This speed ensures the agitation of the magneticparticles 9, while the liquid test medium 8 inside remains relativelystationary with respect to container 3. Switching off the electric motor6 stops rotation of the container 3. The magnetically-induced agitationof the magnetic particles 9 stops and the magnetic particles 9 areattracted to and immobilized at the inside wall of the container 3closest to the magnet 1. At this time, if desired, magnet 1 may be movedcloser to container 3 to tightly aggregate the magnetic particles 9 onthe vertical side of the container 3 to facilitate clean removal of theliquid test medium 8.

FIG. 2 illustrates an alternate preferred embodiment for mixing andseparating magnetic particles according to the present invention whichincludes a test tube 23 removably inserted through an opening in a testtube holder 25 without extending to a rotating support 22. Magnetassembly 21 is adjustably fixed to rotatable support 22 withoutextending to the test tube's bottom end. The magnet assembly 21 may bemoved or fixed at a desired distance with respect to container 23 toadjust the magnetic field strength. The magnet 21 may be either anelectromagnet or a permanent magnet. If the magnet 21 is a permanentmagnet, it is preferably comprised of a rare earth composite such asNeodymium-Iron-Boron with a surface field strength of about 200 Gauss to5 kilo Gauss, sufficient to attract the magnetic particles in the sizerange of about 0.1 μm to 15 μm. The magnet 21 may comprise one or moremagnets of suitable dimensions and geometries so as to define a magneticfield cavity of a desired field strength having a desired cross-sectionwithin the liquid test medium 28 in the test tube 23.

The rotatable disc 22 is mounted to a shaft 24, which is in turnattached to a variable speed electric motor 26. The electric motor 26rotates the magnet 21 orbitally around the vertical axis of thestationary test tube 23 creating an angularly moving magnetic fieldgradient within the test medium 28. The test tube 23 remains motionlesswhile the magnetic field cavity rotates continuously through thestationary test medium 28. The motor 26 may be an electric step motor toprovide a step-wise change of a predetermined distance in the relativeangular position such as described above.

The magnet when rotated continuously is rotated from a resting positionto a speed, preferably between about 50 and 200 rpm in less than onesecond. The angularly moving magnetic field with respect to theaggregating magnetic particles 29 induces the magnetic particles 29 tomove within the magnetic field cavity through the relatively motionlessliquid test medium 28. When the electric motor 6 is switched off, themagnetically induced agitation stops. The magnetic particles 29 in thenow stationary magnetic field are attracted to and immobilized on theinside wall of the test tube 23 closest to the magnet 21. At this time,if desired, the magnet 21 may be moved closer to test tube 23 to tightlyaggregate the magnetic particles 29 on the vertical side of the testtube 23 to facilitates a cleaner removal of the test medium 28.Aggregation of the magnetic particles 28 on the vertical side of thetest tube 23 facilitates removal of the test medium 28 by aspiration orother means.

FIG. 3 illustrates another preferred embodiment of the present inventionfor processing a plurality of test media simultaneously and is a variantof the embodiment of FIG. 2. The apparatus comprises a row of identicaltest tubes 33, fixed in vertical positions by their top ends passingthrough corresponding openings in a fixed horizontal support plate 32.The vertical position of the corresponding row of multiple magnets in amagnet assembly 31 is adjustably fixed without extending to the bottomends of the test tubes 33. The magnet assembly 31 may be moved to andfixed at a desired distance from the test tubes 33 to adjust themagnetic field strength. If permanent magnets are used, they arepreferably of a rare earth type as described above, and are selected tohave suitable dimensions and geometries to define a magnetic fieldcavity with a desired field strength having a desired cross-sectionwithin the liquid test medium 29 in each test tube 33.

A support plate 35 for the magnet assembly 31 is fixed at itsextremities by two shafts 34 a and 34 b. These shafts are eccentricallyattached to pulleys 38 a and 38 b, which are, in turn, connected by adrive belt 39. The pulley 38 a is attached to a variable speed electricmotor 36. The motor 36 rotates the pulleys 38 a and 38 b, therebyimparting an eccentric rotation to support plate 35. This motion causeseach magnet of the magnet assembly 31 to orbit around the vertical axesof its corresponding stationary test tube 33, thereby creating aseparate moving magnetic field gradient within the motionless test media28 of each test tube 33. The motor 36 may be an electric step motor toprovide a step-wise change of a predetermined value in the relativeangular position such as described above.

The magnets when rotated continuously are rotated from a restingposition to a speed, preferably between about 50 and 200 rpm in lessthan one second. The simultaneous movement of multiple magnetic fieldsinduces the aggregating magnetic particles 29 in each test tube 33 tomove within their individual cavities of the magnetic field gradient.Stopping the electric motor 36 stops the rotation of the magnet assembly31 and stops the magnetically induced agitation. The magnetic particles29, in the stationary magnetic fields are attracted to and immobilizedon the inside walls of each test tube 33. If desired, magnet assembly 31may be moved closer to test tubes 33 to tightly aggregate the magneticparticles 29 on the vertical sides of the test tubes 33 to facilitates acleaner removal of the test medium 28. The separation of magneticparticles on the vertical side of the test tubes 33 facilitates removalof the supernatant liquid media by aspiration or other methods.

FIG. 4 illustrates another preferred embodiment of the present inventionfor processing a plurality of test liquid media simultaneously, and is avariant of the embodiment of FIG. 1. The apparatus comprises a row ofmultiple magnets 41, fixed on a support plate 41 b (not shown). Thesupport plate is preferably adjustably mounted to align the row ofmagnets so each magnet corresponds with its respective test tube 43.Support plate 41 b also preferably provides lateral movement to adjustthe distance between the magnet assembly 41 and the row of test tubes43. The magnets 43 thus can be moved to a desired distance from the testtubes 43 to adjust the magnetic field strength. If permanent magnets areemployed, they are preferably a rare earth type as described above andhave dimensions and geometries so as to define a magnetic field cavitywhich accommodates a desired cross-section within the liquid test medium8 in each test tube 43.

The test tubes 43 are removably placed in vertical positions with theirbottom ends resting in a row of shallow grooves on a bottom plate 42. Aportion of their top ends pass through corresponding openings in anupper plate 42 b of the test tube rack 42. The diameter of the openingsin the upper plate 42 b is slightly larger than the diameter of the testtubes 43 so that they can be readily inserted and freely rotated. Theplates 42 and 42 b are spaced apart so as to hold the test tubes 43 in astable vertical orientation.

A drive belt 49 is mounted on two pulleys 48 b and 48 c. Pulley 48 c isattached to a variable speed motor 46, and guided by two parallel rowsof guidance rollers 47 mounted on the top plate 42 b. The guidancerollers 47 are positioned between the rows of openings to slightly pinchthe drive belt 49 so that the drive belt 49 grips the upper ends of thetest tubes 43. Motor 46 moves the drive belt 49. The linear slidingfriction of belt 49 against the external surface of each test tubesimultaneously rotates all test tubes 43 around their vertical axes. Themotor 46 may be an electric step motor to provide a step-wise change ofa predetermined distance in a relative angular position, such asdescribed above.

At a suitable acceleration, test tubes 43 rotate, the relative angularposition of the aggregating magnetic particles 9 in each one of the testtubes 43 and its corresponding magnet 41 is continuously altered. Thisinduces the magnetic particles 9 to move within the cavity of themagnetic field gradient. The test tubes 43 are rotated from restingposition to a speed, preferably between about 50 and 200 rpm in lessthan one second to ensure the agitation of the magnetic particles 9while maintaining the test media 8 inside relatively stationary.Stopping the electric motor 46 stops rotation of test tubes 43 and themagnetically induced agitation. The magnetic particles 9 in each testtube 43 are now attracted to and immobilized at the inside wall closestto the magnets 41. The aggregation of the magnetic particles 9 on thevertical side of the test tubes 43 facilitates removal of the testmedium 8 by aspiration or similar methods. If desired, magnet assembly41 may be moved closer to container 23 to tightly aggregate the magneticparticles 9 on the vertical side of the container 43 to facilitates aclean removal of the test medium 8.

An instrument incorporating the above-described principles of theinvention has been built and is being sold by Sigris Research, Inc.,P.O. Box 968, Brea, Calif. 92622. Literature describing the operation ofthe instrument, specifications and actual performance statistics widelydistributed since 1996 is available from Sigris Research, Inc. and isincorporated herein by reference.

FIG. 12 illustrates another preferred embodiment of the presentinvention for processing a plurality of test liquid mediasimultaneously. It includes a linear drive mechanism mounted on apositioning mechanism and a rotation mechanism. The three mechanismsallow vertical linear movement of a magnet assembly, adjustment of thedistance between the magnet assembly and containers, and rotation of thecontainers. Simultaneous container rotation and linear magnet movementprovides the advantage of processing large volumes of test media with arelatively small quantity of magnetic particles.

The apparatus of FIG. 12 consists of two main parts, linear driveassembly 111 and base assembly 112. Both assemblies are constructed of anonmagnetic material, aluminum being preferred. The linear driveassembly 111 comprises a rigid frame 113 with two fixed guide rods 114and 115 and a centrally located screw shaft 116. The end portions ofscrew 116 are smooth and un-threaded and are mounted in two centrallylocated ends flanges (not shown). The screw 116 is freely rotatable andincludes a roll nut (not shown) which moves linearly in the verticalplane, either up or down, upon rotation of screw 116. A pulley 117 isfixed to the smooth portion of screw 116 protruding from the top plate136 of frame 113 and is connected by a timing belt 118 to another pulley119 fixed to the shaft of a variable speed electric motor 120 mounted onbracket 121 of frame 113. Timing belt 118 is made of neoprene orurethane with precisely formed grooves on the inner side. The belt widthand groove pitch match the dimensions of the teeth on pulleys 117 and119 to provide positive and non-slip power transmission. Suitable timingbelts and gear pulleys may be obtained from Stock Drive Products, NewHyde Park, N.Y. or from other similar vendors.

A carriage 122 is fixed on the roll nut of screw 116. Its verticalmotion is ensured by the accurately aligned guide rods 115 and 114.Linear drive assembly 111 is attached to base assembly 112 by boltingthe bottom plate 139 of frame 113 to a linear slide mechanism 123. A rodwith a knob 128 inserted through a center hole of the base assembly 112is attached to the linear slide mechanism 123. The linear slidemechanism 123 thus can be moved forward or backward by pulling orpushing the knob 28 to position it at a desired distance from thecontainers 124.

A magnet assembly 125 with magnets 126 is removably mounted on thelinear drive carriage 122 by means of three evenly spaced screws 127.This is advantageous because magnets of varying size and geometry can beeasily exchanged. The magnets 126 are aligned with the row of containers124. Their distance from the containers is adjusted by pulling orpushing the knob 128.

The motor 120 rotates the screw 116. The roll nut converts this rotarymotion to a linear motion moving magnet assembly 125 vertically. Thedirection of the linear movement of magnet assembly 125 is controlled bythe clockwise or counter-clockwise rotation of the motor 120 by a motorcontroller. The movement of magnet assembly 125, either upward ordownward can thus be controlled at will and may be repeated for as manycycles as desired.

The position and the stroke length of the linear up and down movement ofthe carriage 122 may be controlled by two position sensors to controlthe lowest and highest extremes of travel of the carriage 125. Anelectronic signal from these sensors may be used to reverse the motorrotation, thereby causing a repeated scanning for a desired length ofthe containers 124 by their corresponding magnets 126.

Electronic motor controllers and position sensor are well known in theart and may be obtained from any one of a number of vendors. Ifpermanent magnets are employed, they are preferably a rare earth type asdescribed above and have suitable dimensions and geometries so as todefine a magnetic field cavity of a desired field strength whichprovides a desired cross-section within the liquid test medium in eachcontainer.

The base assembly 112 includes a mechanism for rotation comprising avariable speed electric motor 129 with a gear pulley 130 fixed to itsshaft. A pulley rotor 131 is attached to each one of a plurality ofholder 134. A timing belt 132 is wrapped around the gear teeth of pulley130 and each of the rotors 131. Although only one rotor 131 is shownnext to a holder 134 for a container 124, it should be understood thateach container holder 134 has a rotor 131 associated with it, which isdriven by the belt 132. The motor 129 and rotor pulleys 130, 131 aresecured in their precise positions by a top metal plate 133 fixed tobase assembly 112. It should be noted that the gear pulley rotors 131are free rotating and their respective shafts protrude fromcorresponding holes in plate 133. The belt width and the inner groovepitch of the timing belt 132 dimensionally match with gear teeth of themotor gear pulley 130 and the rotors 131 to provide positive andnon-slip power transmission. If desired, idling rollers may be installedbetween the pulleys to increase the wrap around the gear teeth for afirmer non-slip power transmission. The motor 129 rotates the timingbelt 132 thereby simultaneously rotating all pulley rotors 131.

Holders 134 are removably mounted on the tapered end of a rotor shaft135 protruding from corresponding holes in plate 133 and provide meansfor firmly holding containers 124 in a substantially vertical position.A removable holder design is advantageous as it provides a convenientmeans to accommodate a variety of container sizes on the apparatus bysimply changing the holders to correspond to the container geometry.

The position of the magnet assembly 125 may be adjusted to a requireddistance from the row of containers 124 by pulling or pushing the knob128. The motor 129 rotates containers 124 around their vertical axes. Ata suitable angular acceleration preferably between about 1.05 to 4.19rads/s², the relative angular position of the aggregating magneticparticles in each container with respect to its corresponding magnet 126is continuously altered, inducing the magnetic particles to mix withinthe cavity of the magnetic field gradient, as described above. While thecontainers 124 are rotating, motor 120 may be switched on to move themagnets 126 up and down in the vertical plane thereby moving themagnetic field cavity in alignment with the vertical axis of thecontainers. Upon reaching a desired length of the container, thedirection of movement of magnet assembly 125 is reversed. This processis repeated for the entire duration of particle mixing necessary to bindthe target species to particle surface by affinity reaction.

It will be recalled that the magnetic particles remain confined in themagnetic field cavity. Particle to target substance ratio therefore maybe adjusted to relatively high levels within the magnetic field cavityto provide reaction conditions, which overwhelmingly favor affinitybinding. By combining a linearly moving magnetic field cavity with theangular movement of particles confined within the magnetic field cavity,a simple and efficient means to process large volumes of test mediawithout a concomitant increase in particle concentration is obtained.This was not heretofore possible.

The motor 129 may be an electric step motor to provide a step-wisechange of a predetermined distance in the relative angular position suchas described above. Similarly, motor 120 may be an electric step motorto provide a step-wise change of a predetermined distance in thevertical plane. Various combinations of continuous and step-movement forthe rotation and linear movement may be utilized. In every case theoptimum speed of rotation and linear movement will be determined bytrial and error.

For separation, the linear drive motor 120 is turned off. The magnetassembly 125 is brought to a home position. The rotation drive motor 23is turned off. The magnetic particles in the containers 124 areattracted to and immobilized at the inside wall closest to the magnets126. The aggregation of the magnetic particles on the vertical side ofthe container 124 facilitates removal of the test medium by aspirationor similar methods. If desired, magnet assembly 125 may be moved closerto containers 124 by moving knob 128. This tightly aggregates themagnetic particles on the walls of the containers 124 to facilitate aclean removal of the test medium.

FIGS. 5 a through 5 f illustrate the preferred steps in a methodpracticed by the preferred embodiments described above, using affinityreactive magnetic particles of about 2.8 μm for the purpose ofbioassays, or for the isolation of cellular or molecular species from asample solution or suspension of biological fluids.

FIG. 5 a shows an apparatus of FIG. 2, in which a suspension of magneticparticles 58 in a sample solution is dispensed with a pipette 59 c intoa test tube 23 of about 10 mm diameter. A magnet 21 of about 45 MGOe ismoved to a distance of about 5 mm from test tube 23 so as to create afield of about 600 Gauss in the center of the test tube. This preferreddistance is determined by measuring the magnetic field inside the tubeby a magnetometer. The motor is turned on and the magnetic particles 58are mixed by rotating the magnet 21 around the test tube 23. FIG. 5 bshows the same apparatus when mixing is completed, rotation of themagnet 21 has stopped, and the magnet is moved closer to the test tube23. The magnetic particles 58 are immobilized against the inner wall oftest tube 23 closest to the stationary magnet 21.

FIG. 5 c shows the apparatus during a washing step. In this step, anoutlet tube 59 a aspirates the supernatant test medium and an inlet tube59 b adds a suitable wash solution into the test tube 23. The magneticparticles 58 are then mixed in the wash solution. The old wash solutionis aspirated and new clean solution may be added. The washing step maybe repeated as many times as required.

FIG. 5 d shows the apparatus stopped for the addition of one or morereagent solutions by pipette 59 c for effecting a desired analyticalreaction for a bioassay or a chemical displacement reaction to elute thetarget substance from the magnetic particles 58.

FIG. 5 e shows the same apparatus turned on for dispersing and mixingthe magnetic particles 58 for carrying out the desired reaction.

FIG. 5 f shows the apparatus stopped to separate the magnetic particles58 from the reaction medium. In the case of bioassays, the supernatantliquid may be measured by any desired measurement method, eitherdirectly in test tube 23 or by transferring it elsewhere. For thepurpose of isolating a cellular or molecular species, the supernatantmay be transferred to a suitable container for subsequent treatment asdesired. Examples of actual separations of mRNA and protein aredescribed in a technical brochure entitled “MixSep”, obtainable fromSigris Research, Inc., and are incorporated herein in its entirety.

Various preferred configurations of magnet assemblies and their positionwith respect to a container will now be described with reference toFIGS. 6 through 9. FIG. 6 shows a perspective view of an embodiment ofthe magnet assembly 61 according to the invention wherein a rectangularpermanent magnet 62 is fixed on a nonmagnetic base 63 and placed inproximity to a container 64 to generate a cavity of magnetic fieldgradient 65 in a cross-section of a liquid test medium 66. The usablemagnetic field remains mostly confined within this cavity, i.e., thereis negligible field strength outside the cavity.

FIG. 7 shows two magnet assemblies, 71 a, 71 b, each comprised of tworectangular permanent magnets 72 a and 72 b fixed on two nonmagneticbases 73 a and 73 b, respectively. The two magnet assemblies 71 a, 71 bare located on the opposite sides of a container 74 with similarmagnetic poles facing each other to distort the magnetic flux lines andgenerate a cavity of magnetic field gradient 75 in the liquid testmedium 76 and two loci of magnetic force in the cavity 75 as explainedabove (see FIG. 11 a). Such an arrangement may be particularly effectivefor mixing magnetic particles.

FIG. 8 shows a magnet assembly 81 designed to generate multiple cavitiesof magnetic field gradient in a container 84. An array of sixrectangular permanent magnets 82 a to 82 f fixed on a nonmagneticsupport frame 83 is preferred. Magnets 82 a to 82 f are verticallymounted on the non-magnetic support 83 wherein each magnet issubstantially separated by a non-magnetic spacer and like poles overlike poles so that magnetic flux lines from each magnet traversing thetest medium 86 are mutually repulsive and generate a plurality ofdistinct magnetic field cavities. The spacing between magnets should besuch as to prevent the intermixing of magnetic particles from one fieldcavity to other. Such spacing may be even or uneven.

The magnet assembly 81 is placed at a desired distance from thecontainer 84 to generate six separate cavities of magnetic fieldgradient 85 a to 85 f in a liquid test medium 86. Such multiple magneticfield cavities are useful for isolating a multiple of target substancesfrom a test medium in a single operation. The affinity magneticparticles in a given cavity will specifically bind a given targetsubstance only. Specific types of magnetic particles are addedsequentially from bottom cavity to top cavity. In the first step, thecontainer is filled with a suspending solution to the level of the firstcavity, magnetic particles are then added and allowed to aggregate. Thisstep is repeated until all cavities are filled with the desired type ofmagnetic particles. The suspending solution is then removed and thecontainer filled with the test medium. Alternatively, a test liquidsample may be layered over the test medium and the target substanceallowed to settle down by gravitational force while the particles aremixing. Such a method is of particular use for isolating differentcellular components in a single process. Mixing and separation are thencarried out as described in connection with FIG. 5.

FIG. 9 shows two magnet assemblies 91 a and 91 b, each comprising anarray of six evenly-spaced rectangular permanent magnets 92 a to 92 ffixed on two nonmagnetic support frames 93 a and 93 b, respectively. Thespatial and pole arrangements of assemblies 91 a and 91 b are similar tothe one described in FIG. 8. The two magnet assemblies 91 a and 91 b arelocated on the opposite sides of a container 94 with like magnetic polesfacing each other. Six cavities of magnetic field gradient 95 a to 95 fthus generated in a test medium 96 by distorted magnetic flux lines oftwo operative magnetic fields in each cavity.

The various configurations of magnet assemblies and position asdescribed above may be advantageously employed in the embodiments of theinvention depicted in FIGS. 1 to 4 and 12.

As mentioned above, permanent magnets and electromagnets areinterchangeable in most configurations of the present invention.However, those configurations that require movement of a magnet are moreeasily realized with permanent magnets. Electromagnets requirecommutators or other arrangements to conduct electricity to the movingmagnets. There are certain unique configurations in which electromagnetsare greatly preferred. FIG. 10 a shows two electromagnet coils 101 a and101 b mounted on a support frame 104 and displaced at about 180 degreesat the exterior of a container 102 with the liquid test medium andmagnetic particles 103 inside. FIG. 10 b shows a cross-section of asingle container 102 with the liquid test medium and magnetic particles103 surrounded by a ring of individual electromagnet coils 101 a to 101r mounted on a support frame 104.

Here neither the container 102 nor the electromagnets 101 actually move.Instead, angular movement is induced in the magnetic particles suspendedwithin the test medium 103 inside the container 102 by sequentiallyenergizing the electromagnets. This sequential energization may be“binary” (i.e., on and off) or “analog,” in which a first electromagnetis gradually fully energized, and then has its power reduced, while thenext electromagnet is gradually energized, and so on. It will beapparent that rate of motion of the magnetic particles 103 can bemodulated by the rate of change and the degree of overlap between thesequential electromagnets.

The exact number of sequential electromagnets employed will depend onthe size of the container 102 and other parameters. FIG. 10 a shows thatthis configuration reduces to a configuration not unlike that of FIG. 7,but with two opposed electromagnets rather than two permanent magnets.The angular movement from one magnet to the other in its simplest formis 180 degrees so that the magnetic particles in the test medium 103will move in relatively straight lines back and forth across thecontainer 102. More variety is preferably added to the paths of themagnetic particles by modulating the polarity, as well as the powerlevel of the electric current, thereby altering the direction of themagnetic poles with alterations of the magnetic field corresponding tothose shown in FIGS. 11 a and 11 b.

It has been found that a configuration employing four electromagnetsequally spaced (i.e., 90 degrees apart) around a container can producevery acceptable agitation of magnetic particles through a judicious useof sequential activation of the electromagnets and through polarityreversals, as discussed above.

The container defining the mixing and separation chamber includes atleast one opening for the addition and removal of a test medium. Thecontainer is preferably of substantially cylindrical form and made froma magnetically permeable material such as plastic or glass.Additionally, the inside surface of the chamber may be biocompatibleand, if desired, the chamber may be sterilized for aseptic processing ofthe test media. The volume of the container is not critical as long asan adequate magnetic field gradient can be provided to accommodate thechamber and, particularly, can accommodate the desired cross-section ofthe liquid test medium inside.

As shown in FIGS. 1 through 9 and 12, the container used to hold thetest medium may be a test tube or an eppendorf type of tube with aconical bottom. The volumetric capacity of the test tube is preferablybetween 250 μl to about 18 ml as usually employed in researchlaboratories. The various configurations of apparatus as described abovecan be easily scaled up to process much larger volumes of liquid testmedia as may be required for clinical applications. In all cases, thesize and geometry of the magnet is adjusted to generate an adequatemagnetic field strength within the field cavity of the test mediuminside a particular size of container.

Although embodiments of the present invention particularly suited foruse in the research laboratory preferably employ readily removable andreplaceable containers such as test tubes, diagnostic and other devicesemploying the teachings of the present invention might employ permanentflow cells or other nonremovable chambers for mixing and separation.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described preferred embodiment can beconfigured without departing from the scope and spirit of the inventionwherein the affinity reactive magnetic particles are admixed with theliquid test medium in a container by effecting a relative angularmovement of the magnetic particles in the liquid test medium, while theliquid remains essentially motionless. Although relative angularmovement is achieved by rotating the container or orbiting the magnet,alternative mechanism will be obvious to a skilled artisan. For example,relative angular movement between the magnetic source and theaggregating magnetic particles may be effected by moving the magnet orthe container by a linear motion mechanism using an appropriate linearacceleration. For this purpose, at least two magnets will be positioneddiametrically opposite one another relative to the container butstaggered so that the magnetic field inside the container generated byone magnet is substantially unaffected by the magnetic field generatedby the other magnet. The linear acceleration of either the magnet orcontainer will mix the magnetic particles in a manner analogous to the180° step rotation movement described earlier.

FIGS. 13-15 shows other preferred embodiments of the present inventionwhich generally include a “sample plate” with apertures for holding thecontainers, and a “magnet plate” with at least two arrays of magnetsmounted onto it, and a “base plate” with at least 2 “side plates”supporting the “sample plate” and “magnet plate”.

The magnet arrays in either a circular or rectangular pattern aremounted on the “magnet plate”. The change in the relative angularposition between the magnetic particles in the liquid sample in acontainer is preferably effected in a single step movement at a suitableacceleration by an electric step motor. Either the sample plate or themagnet plate can be moved and such motion linear or rotary so that eachsuccessive movement brings the opposite lateral sides of the containerin front of the staggered magnets. Such an angular change between thecontainer and magnet brings about the re-suspension and mixing of themagnetic particles in the liquid sample in a manner analogous to the180° step rotation movement described earlier. In the absence of suchmovement, the magnetic particles in the liquid sample move to lateralside inside the container nearest to the magnet and thus separated fromthe liquid sample.

The devices according to the invention are advantageous for processinglarge number of samples and of great utility in molecular diagnostics,forensic DNA analysis and molecular biology fields.

FIGS. 13A-C shows various isometric views of a device with the four sideplates covering the device removed. It includes a base plate 131, anelectric motor 137, preferably a step motor, a chuck 136 mounted on themotor shaft 137A, a magnet plate 132 with plurality of magnets 134mounted in a circular pattern, a round shape sample plate 133 with a hasplurality of apertures for inserting containers 135 with the magneticparticles in a liquid sample. Hereafter container shall mean containerwith a suitable volume of a liquid sample containing magnetic particles.Disposable plastic test tubes such as Eppendorf type of tubes arepreferred but other type can also be used.

Sample plate 131 includes 2 holes 133B, which tightly fits into thealigning dowels 136B on the chuck 135 to align the four holes 133C withthe four threaded holes 136A of the chuck through which four threadedscrews are used to fasten sample plate 131 to chuck 136. Sample platecan be rotated by motor 137, preferably at predetermined angularpositions in successive steps preferably with a delay time between eachstep. The electric motor will be driven by appropriate electronic motioncontrollers, available through many vendors such as Nyden Company,Fremont, Calif.

The magnet plate 132 is attached to the top frame of motor 137 throughfour holes 135 by long fasteners screwed to the base plate 131 so thatboth the motor 137 and magnet plate 132 remain firmly secured. Themagnet plate 132 has a plurality of magnets 134 mounted in two circulararrays, which are concentric. The circular magnet arrays aresufficiently spaced apart to allow the free rotation of the containers135 between them.

Permanent magnets of rectangular shape are preferred but other shapessuch as cylindrical can also be used. High-energy magnets of the NdFeBtype are particularly preferred. The magnets 134 will be of appropriatesize (width and length) to provide sufficient magnetic field strengthover substantial part of the lateral surface area of the container 135.The arrangement where similar poles such as north-north or south-southare diametrically opposite one another relative to the container ispreferred. As shown in FIG. 13B, the individual magnets 134 in the twocircular arrays are positioned diametrically opposite one anotherrelative to the container but staggered so that the magnetic fieldinside the container generated by one magnet is substantially unaffectedby the magnetic field generated by the other magnet.

For example, in a device for Eppendorf type of container, the magnets ofthe size about 10×10×25.4 mm was used in two circular arrays. Whenassembled (FIG. 13A), the distance between the magnet plate 132 and thesample plate was adjusted to about 11-17 mm distance so that thecontainer rotates without hindrance.

Sample plate 133 was rotated by the step motor 137 in steps of about30°, which positions the container in successive steps in front of themagnets, which are diametrically opposite side of the container. Thisbrings about an 180° change in the direction of magnetic fieldattracting magnetic particles in the liquid sample. Continuing thisstep-wise rotation suspended and mixed the magnetic particles. Onstopping the rotation, the container remained stationary in front of themagnet resulting in the separation of the magnetic particles from theliquid sample. A 15° step move brought the container at an intermediateposition between the magnets to substantially remove the effect ofmagnetic field on the container which was useful in some downstreamapplication such as eluting the affinity bound nucleic acids from themagnetic particles.

Although rotary motion of the sample plate has been described, it isobvious that the sample plate can be fixed while the magnet plate isrotated and such a configuration may useful in certain situations.

FIGS. 14 A-D shows various isometric views of a device with a lineararray magnet mounted in assemblies arranged in of plurality of rows overthe magnet plate and parallel over it a sample plate with plurality ofapertures for holding the containers.

It includes a base plate 141 with two parallel side plates 142A-B withrectangular grooves 142G mounted thereon, a magnet plate 143 with ninemagnet assembles mounted in equidistance rows with assembly 146Aalternating with respect assembly 146B and a sample plate 144 fixed overthe top of side plates 142A-B and a sample plate 144 mounted over theside plates 142A-B and has an 8×12 array of apertures 144A for insertingthe containers 145 containing magnetic particles in liquid samples. Asshown in FIG. 14B, the left and right sides of magnet plate 143 can beinserted in the groove 142G of the two parallel side plates 142A-B toprovide a guided linear sliding movement of the magnet plate parallel tothe sample plate 144. The magnet plate 143 can be mechanically attachedto a linear electric step motor, linear actuator or other suitablemotorized mechanical means to horizontally move the magnet plate withinthe linear guide of the two grooves 142G.

The magnet assemblies 146A-B comprises of magnets 147 fixed in evenlyspaced slots of a nonmagnetic harness by an adhesive or suitablemechanical means with seven magnets in 146A and six magnets in 146B.Permanent magnets are preferred, particularly high energy magnets ofNdFeB type. The magnets 147 will be of appropriate size (width andlength) to provide sufficient magnetic field strength over substantialpart of the surface area of the container 146. The arrangement wheresimilar poles are diametrically opposite one another relative to thecontainer is preferred.

As shown in FIG. 14C, the magnet plate 143 has a plurality of magnetassemblies mounted equally spaced and parallel rows with magnet assembly146A alternating magnet assembly 146B. The separation distance betweenthe assemblies should be sufficient to allow a hindrance free movementof magnet assemblies 146A-B with respect to the containers 145 betweenthem.

FIG. 14D is a partial cut of the top view of the invention showing theapertures 133A of the sample plate are positioned at the center betweenmagnet assemblies 146A-B. It will be recalled that the containers 145containing magnetic particles in a liquid sample would be insertedthrough the apertures 133A in the sample plate. The positions of thecontainers will therefore be identical to the positions of apertures.Referring to FIG. 14D, it can be seen that individual containers in eachrow are precisely positioned in front of the alternating individualmagnets 147 in the magnet assembly 146A and magnet assembly 146B. Thedirection of the magnetic field acting on the lateral side of thecontainers alternately reverses. It should be noted that magnetic fieldgenerated by one magnet is substantially unaffected by the magneticfield generated by the other magnet.

When magnet plate 143 is stationary with magnets and the samplecontainers in their respective arrays aligned, magnetic particles insidethe containers will collect on the lateral wall of the containersnearest to its respective magnets. A linear step movement of the magnetplate 143 will position the magnets at the diametrically opposite sideof the containers thereby attracting the magnetic particles to aposition opposite to previous. Repeated forward and backward step-wisemotion of the magnet plate will cause the suspension and mixing ofmagnetic particles in the liquid sample and when this motion is stoppedseparation of magnetic particles.

The invention shown in FIG. 14A, can process 96 Eppendorf types ofcontainers simultaneously. It contains the permanent magnets of about10×10×25.4 mm size which are mounted in the evenly spaced of about 50.8mm slots in the magnet assembly with seven magnets in magnet assembly146A and six magnets in 146B. Nine rows of magnet assemblies weremounted on the magnet plate 143 with five of assembly 146A alternatingwith four of assembly 146B. A distance of about 12 mm separated the rowsof assemblies. When assembled, the distance between the magnet plate 143and the sample plate 144 is adjusted to so that Eppendorf samplecontainer 145 is maximally exposed to the magnets 143 and the magnetplate 143 moves without hindrance.

The magnet plate was mechanically connected to a linear step motor tohorizontally move the magnet plate 143 to about 25.4 mm lateral distancemm in the forward and backward directions. A delay time of about 0.5 to60 seconds was imposed during each stroke of the linear movement. Such amotion profile was electronically controlled and repeatedly positionedthe magnets at the diametrically opposite side of the container whichsuspended and mixed the magnetic particles inside the Eppendorf untilthe motor was stopped to separate the magnetic particles from the liquidsamples.

Although linear motion of the magnet plate is described, it is obviousthat the magnet plate can be fixed while the sample plate ishorizontally moved to repeatedly position the sample container betweenthe diametrically opposed magnets.

FIG. 15 shows an isometric view of an apparatus for mixing andseparating magnetic particles in the wells of a 96-well microplate. FIG.15A is an exploded isometric view corresponding to FIG. 15 and shows abase plate 150 with four guide rods 150 A, two magnet assemblies 151-152with magnets 155 mounted in linear arrays thereon, a sample plate 153with apertures for inserting the 96 wells 154A of the microplate 154.

The disposable plastic microplates 154 known as “skirt-less” type arewidely available and have integrally formed multiple wells for holdingliquid samples. As shown in FIG. 15A, the wells 154A in the microplate154 are closely spaced and arranged in an 8 by 12 array. The volumetriccapacity of the wells is usually in the range of 250-350 micro liters.While a ninety-six well microplate is shown, this invention is equallyapplicable to standard 6, 12, 24, 48 tissue culture plates as well as96-well microplates with a volumetric capacity of 1-2 ml.

The magnet 155 are preferably of cylindrical shape and the high energymagnets of NdFeB type in order to provide strong magnetic fieldsadjacent to wells in microplate 154.

The magnet assemblies 151 and 152 are slotted in the shape of “fingers”in order to permit the unhindered independent movement of either magnetassembly in the vertical direction to place the magnets between thewells of microplate. A rectangular plate with appropriate access holesfor magnet passage can also be used instead of the slotted shape formagnet assemblies.

The magnet assembly 151 includes two guide holes 151A and a 4×6 array ofindividual rod-shaped magnets mounted on the four slots of the assembly.The magnet assembly 151 is precisely positioned by inserting it throughthe guide holes 151A in the two individual guide rods 150A mounted onthe right of the base plate 150. The magnet assembly 152 include twoguide holes 152A and a 5×7 array of individual rod-shaped magnetsmounted on the five slots of the assembly. The magnet assembly 152 issimilarly positioned by inserting it through the guide holes 152A in thetwo individual guide rods 150A mounted on left of the base plate 150.Both magnet assembles can be independently moved up towards the bottomof microplate 154 through the linear guide provide by the holes androds. A suitable roller bearing may be used in the holes to provide arigid mechanical support to assemblies as well as friction lessmovement. Each assembly can be mechanically attached to a linearelectric step motor, linear actuator or other suitable motorizedmechanical means to provide independent movement to each assembly. FIG.15B shows a front view corresponding to FIG. 15A, illustrating therelative positions of the mounted magnets 155 in the assemblies withrespect to wells micro plate. The gaps between slots of the assembly 152(hidden) allow the positioning of mounted magnets of the assembly 151between the well of the micro plate 154.

The sample plate 153 has a plurality of apertures 154 A arranged as 8×12array of individual apertures corresponding to 8×12 array of individualwells of the microplate through which the wells can be inserted andprecisely positioned with respect to magnets 155 mounted on the twomagnet assemblies 151 and 152. The sample plate 153 is fixed at the topof four guide rods 150A by means of threaded fasteners. The micro platethus remains fixed and the wells precisely positioned to permit accessof mounted magnets between the exterior spaces of the wells underneaththe microplate 154. Sufficient distance between the base plate 150 andthe sample plate so that magnetic field of the mounted magnets 155acting on the wells of micro plate is negligible. Magnetic field becomesactive only when the magnet assembly is moved upward and the mountedmagnets positioned between the wells of the micro plate 154 and near thesample plate surface underneath.

FIG. 15C shows the relative positions of the 4×6 array of magnets andthe 8×12 array of apertures 153A when the magnet assembly 151 upwardnear the sample plate 153. Apertures can be assumed to represent thewells, as the 8×12 array patterns of both the sample plate 153 and microplate 154 are identical. Referring to FIG. 15C it can be seen that the24 magnets are uniformly distributed within the boundaries of 96 wellsand the radial magnetic field of each individual magnet essentially actson four surrounding wells. Magnet positions in each row and column aresufficiently apart so that the effect of their magnetic fields on thedistant wells can be considered negligible. Magnetic particles in theliquid sample in each set of four wells will move and aggregate on thelateral surfaces inside the wells nearest to the magnets.

FIG. 15D shows the relative positions of the 5×7 array of magnets andthe 8×12 array of apertures 153A when the magnet assembly 151 is moveddown and the magnet assembly 152 is moved upward near the sample plate153. As previously, the apertures can be assumed to represent the wells.

A comparison between FIGS. 15C and D Figure, reveals that the magnets inthe magnet assemblies 151 and 152 are positioned diametrically oppositeone another relative to the wells of microplate 154 and the radialmagnetic field of each individual magnet will now attract the magneticparticles in the liquid sample in each well on the diametricallyopposite lateral surface inside each well.

Successive rapid upward movement of one magnet assembly at a time whilethe other magnetic assembly is rapidly moved down will bring about themixing of magnetic particles in the liquid sample in each well in asimilar manner as described earlier. For the separation of magneticparticles in the wells either one or both magnetic assemblies may bemoved upward and made stationary till all the particles have separatedin the wells, which permits the removal of the supernatant liquid.

FIG. 16A shows an isometric view of a device for single containerpreferably a container of large volumetric capacity such as 50 ml. Itincludes a magnet assembly comprised of two hexagonal magnet plates 161and 162, each with a mounted magnet 166. Rare earth high-energy magnetsare preferable and appropriate dimensions to provide maximum magneticfield area to cover the lateral part of the container 165. Both themagnet plates are fixed to the shaft 163 in parallel coaxial positionswith the two magnets opposite each other and separated by an angularposition of 90° and preferably by an angle of 180°. Other angularpositions may be used provided that magnetic field generated by onemagnet is substantially unaffected by the magnetic field generated bythe other magnet. Although rectangular magnets are preferred, round orother suitable shapes for magnets can also be used. Similarly, shapesother than hexagonal can also be used for magnet plates.

The shaft 163 can be the shaft of a step motor to directly rotate themagnet plates or a separate cylindrical shape rod, which can bereversibly attached to a step motor shaft with a suitable shaft couplersuch as Fairloc Shaft Coupling available from Stock Drive Products, N.Y.

The sample plate 166 includes an aperture for inserting the container165 and is attached to a height adjustable clamp of a laboratory standto permits the positioning of the container 165 at the center of the twomagnet plates without hindering its rotary movement of the magnet platesas shown in FIG. 16B. When the shaft is rotated, the two coaxial magnetplates rotate simultaneously which successively brings each mountedmagnet at the opposite lateral sides of the container. Step motion of apredetermined angle by step motor with an imposed delay time of about0.5 second between each step is preferred to suspend and mix themagnetic particles in the sample liquid of the container and by stoppingthis rotation and positioning one of the magnet adjacent to thecontainer separates magnetic particles from the liquid sample on thelateral wall inside the container to allow the removal of thesupernatant liquid.

The relative angular movement is induced in the magnetic particles byeither rotating a magnetic field around a stationary container orrotating the container relative to an immobile magnetic field. Themagnet creating the field is disposed outside the container and definesa cavity of magnetic field gradient within the liquid test medium. Anycontainer configuration may be utilized, such as, for example, adoughnut-shaped container. In such a container the magnetic source maybe “outside” of the container and “within” the container, if it occupiesthe hole of the doughnut. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practicedotherwise than as specifically described herein.

The following examples further describe in detail the manner and processof using the present invention. The examples are to be considered asillustrative but not as limiting of this invention. All manipulationsgiven in the examples are at ambient temperature unless otherwiseindicated.

EXAMPLE 1

The effect of angular acceleration on magnetic particle mixing wasdetermined by using a device similar to the apparatus shown in FIG. 12except that the linear drive mechanism for vertical movement of themagnet assembly was switched off. The magnet assembly included six rareearth type (NdFeB) permanent magnets of about 35 MGOe. The electricmotor (FIG. 12, 129) was a two phase stepping motor driven by a computerprogrammable controller-driver. The rotary motion of the stepper motorresponds to sequence of digital pulses from the controller-driver. Theangular acceleration is directly related to the frequency of inputpulses and the length of rotation is directly related to number ofpulses applied. A 1.6 ml microcentrifuge tube having diameter of about10.6 mm was used as a container. A 250 μl suspension of about 50 millionparamagnetic beads in phosphate buffer saline, pH 7.5 containing 2.5%bovine serum albumin was transferred to a microcentrifuge tube. Thebeads were obtained from Dynal, Inc., and their reported physicalcharacteristics were: spherical and uniform size of 4.5 μm, magneticmass susceptibility of about 16×10-5 m3/kg. The microcentrifuge tube wasplaced in the holder (FIG. 12, 134) and the magnetic field acting insideof the microcentrifuge tube was adjusted by the knob (FIG. 12, 128) sothat the suspended beads aggregate within 60 seconds at the inside wallclosest to the magnet assembly. The magnetic field gradient measuredinside the container varied from about 1.1 kGuass (closest to magnet) toabout 0.4 kGuass (farthest from magnet). Once the magnetic beads hadaggregated, the microcentrifuge tube was accelerated from rest to apreselected speed (rpm) within one second time. The angular accelerationwas calculated from the following formula:α=(ω₁−ω₀)/t=rad/s²

Where α angular acceleration (rad/s²), ω₁ is angular velocity in radiansper second after one second, ω0 angular velocity in radians per secondat rest which in this instance is zero, and t is the time in secondwhich in this instance is one second. The mixing efficiency of the beadsat various acceleration rate was estimated by observing the mass ofbeads remaining aggregated as well as the cloudiness of the suspension(scale of + to 5+). Continuous rotation was used for speeds between 5 to200 rpm. The effect of acceleration on mixing efficiency could beclearly observed when tube was accelerated from rest to an angularposition of 180° in a singe step and stopped. Although a microscope maybe used but a visual examination was also adequate. The results areshown in Table 1. TABLE 1 Speed ω₁ α (rpm) (rads/s) (rads/s²) MixingEfficiency  5 0.52 0.10 No Mixing, beads remain substantially aggregated10 1.05 0.21 No Mixing, beads remain substantially aggregated 40 4.190.84 Cloudiness +, about 90% beads remain aggregated 50 5.24 1.05Cloudiness ++, about 80% beads remain aggregated 100  10.47 2.09Cloudiness +++, about 30% beads remain aggregated 200  20.94 4.19Cloudiness +++++, less than 5% beads remain aggregated 180° Step 209.4441.89 100% mixing

EXAMPLE 2

The effect of angular acceleration on purification efficiency wasdetermined by isolating genomic DNA from human whole blood. The basicexperimental set up was as described in Example 1 and angularacceleration of 0.1, 0.21 and 4.19 rads/s2 was used. The experimentsconsisted of three identical isolations using EDTA anticoagulated bloodand magnetic beads from Dynabeads DNA Direct kit (commercially availablefrom Dynal, Inc., Lake Success, N.Y. 11042). The process of DNAisolation in this kit relies upon cell lysis to release the DNA, whichis then adsorbed at the surface of the beads. It was assumed that mixingefficiency would be directly reflected by comparing the yields of DNAisolated at the three angular acceleration. A 200 μl suspension of beadsfrom the kit was pipetted in a siliconized microcentrifuge tube. Themicrocentrifuge tube was then placed in the tube holder of the apparatusand the magnetic field acting inside of the tube was adjusted by theknob (FIG. 12, 128) so that the suspended beads aggregate within 60seconds at the inside wall closest to the magnet assembly. Once thebeads had aggregated, a 10 μl of EDTA anticoagulated blood was added tothe clear solution inside the tube. The microcentrifuge tube wasaccelerated from rest to a preselected speed (rpm) and the rotation ofthe tube was continued for about five minutes. During this rotation, thebeads, if mixing, would adsorb DNA. The rotation was then stopped andthe magnetic field acting inside of the tube was increased to maximum bybringing the magnet assembly closest to the tube holder by adjusting theknob (FIG. 12, 128). The beads aggregated at the inside wall closest tothe magnet assembly and the supernatant was withdrawn. The beads werethen washed twice with 200 μl of the wash buffer of the kit. During eachwashing the beads were mixed in the washing buffer by rotating the tubefor two minutes at the angular acceleration used for DNA adsorption. Thesteps for the separation of beads and removal of supernatant wash bufferwere as described earlier. The tube was then removed from the apparatusand bead/DNA complex resuspended in 50 μl of resuspension buffer of thekit (10 mM Tris HCl, pH 8.0) by pipetting up and down 30-40 times untilthe suspension is homogenous. DNA was then eluted by incubating the tubeat 65 Co for five minutes. The tube was then placed back on theapparatus to separate the beads and the supernatant containing theeluted DNA was transferred to a clean tube. The DNA content in thesupernatant was then determined by measuring optical density (OD) at 260and 280 nm. The ratio OD₂₆₀/OD₂₈₀ of 1.7 indicated that the isolated DNAwas pure. The OD260 was then used to calculate the concentration of DNA.This technique for the determination of DNA is well known and widelyused in the molecular biology art. The yields of genomic DNA isolated atangular acceleration of 0.1, 0.21 and 4.19 rads/s2 are shown in Table 2.TABLE 2 Speed α DNA Yield (rpm) (rads/s²) Mixing Efficiency ng 5 0.10Aggregated beads roll over one Un-detectable another 10 0.21 Aggregatedbeads roll over one Un-detectable another 200 4.19 Beads are dispersedand mixed 250

1. Apparatus for mixing and separating magnetic particles in a pluralityof containers with liquid and said particles therein, said apparatuscomprising: a. first support means for holding said containers insubstantially straight parallel rows which are spaced apart; b. secondsupport means for holding magnets in substantially straight parallelrows separated by sufficient distance to place said rows of containersthere between to dispose magnets alternately on opposite sides of eachcontainer in close proximity wherein said rows of magnets are mounted onat least two separate supports which are independently moveablesubstantially upwardly or downwardly to alternately position saidmagnets on the opposite sides of said containers rows; c. means forproviding an oscillating movement to said two independent supportssuccessively in substantially linear path in the vertical plane todispose said magnets alternately on opposite sides of each container todisperse said magnetic particles in liquid inside said containers; andd. means for stopping said motion in vertical plane and moving said twoindependent supports to position said magnets in said magnet rows inclose proximity to each container of said container rows to aggregatesaid magnetic particles on the inner wall of said containers.
 2. Theapparatus of claim 1 wherein acceleration of movements in vertical planeis at a rate of about 0.2 m/s² to about 1 m/s² with a delay time ofabout 0.25 s to about 120 s or over 120 seconds between successivemovements of said two support means.
 3. The apparatus of claim 2 whereinsaid oscillating movement is started and stopped by at least oneelectric motor operated and controlled by electrical means.
 4. Theapparatus of claim 1 wherein said magnetic particles are non-colloidalparamagnetic or super paramagnetic.
 5. The apparatus of claim 1 whereinsaid containers is a 96-well microplate held on a support means and oneof said two independently movable supports carries a 6×4 array of evenlyspaced magnets and the other of said two independently movable supportscarries a 7×5 array of evenly spaced magnets wherein alternateoscillating movement of said two independently movable supportsalternately positions their respective magnets on the opposite sides ofwells of said 96-well microplate.
 6. The apparatus of claim 1constructed and arranged to purify molecular and cellular entities byaffinity binding on magnetic, paramagnetic or super paramagneticparticles and separation of said magnetic particles from liquid bymagnets.
 7. Apparatus for mixing and separating magnetic particles incontainers with liquid and said particles therein comprising; a. a firstsupport means for mounting one or more containers in a ringconfiguration; b. a second support means for mounting magnets in atleast two concentric magnet rings spaced apart to position saidcontainer ring there between; c. said first support means and saidsecond support means aligned in the apparatus to make said containerring and said two magnet rings concentric on a common axis; d. said twomagnet rings constructed and configured to dispose a magnet of each ringalternately on opposites sides each container; e. means for providingrotary motion to either said first support or said second support in thehorizontal plane to alternately displace each magnet of said magnetrings at opposite sides of each container in said container ring todisperse said magnetic particles in liquid inside said containers; andf. means for stopping said rotary motion to position each magnet in saidtwo magnet rings in close proximity to each container of said containerring to aggregate said particles on the inner wall of said containers.8. The apparatus of claim 7 wherein the said magnets are permanentmagnets and magnets in said two concentric rings have similar polesopposite one another.
 9. The apparatus of claim 7 wherein said rotarymotion is started and stopped by at least one electric motor operatedand controlled by electrical means.
 10. The apparatus of claim 7 whereinsaid magnetic particles are non-colloidal paramagnetic or superparamagnetic.
 11. The apparatus of claim 9 wherein said rotary motion isstepwise angular displacement at an angular acceleration of about 21rads/s² or higher.
 12. The apparatus of claim 11 wherein said stepwiserotary motion has a delay time of about 0.25 seconds to 120 seconds, andover 120 seconds between successive steps.
 13. The apparatus of claim 11wherein said stepwise rotary motion is in steps of about 10 degrees to30 degrees and unidirectional or bidirectional oscillating betweenclockwise or anticlockwise directions.
 14. The apparatus of claim 7constructed and arranged to purify molecular and cellular entities byaffinity binding on magnetic, paramagnetic or super paramagneticparticles and separation of said magnetic particles from liquid bymagnets.
 15. Apparatus for mixing and separating magnetic particlescomprising: a. at least one said container held in a fixed uprightposition in the said first support; and b. at least two magnets mountedin orthogonal positions on two separate second supports facing eachother and sufficiently spaced to position said container in the middle;c. said two second supports rotatable in unison on a common axis by tosuccessively dispose magnets on the opposite sides of said container;and d. said container and the said two second supports being orientatedin substantially vertical orientation.
 16. The apparatus of claim 15wherein said rotary motion is by at least one electric motor operatedand controlled by electronic means.
 17. The apparatus of claim 15wherein said magnets are separated by an angular position of 180 degreesor less.
 18. The apparatus of claim 15 wherein said rotary motion isstepwise at an angular acceleration of about 21 rads/s² or higher with adelay time of about 0.25 seconds to 120 seconds, and over 120 secondsbetween successive steps.
 19. The apparatus of claim 15 wherein saidmagnetic particles are non-colloidal paramagnetic or super paramagnetic.20. The apparatus of claim 15 constructed and arranged to purifymolecular and cellular entities by affinity binding on magnetic,paramagnetic or super paramagnetic particles and separation of saidmagnetic particles from liquid by magnets.